Negative effects of phosphorus addition outweigh effects of arbuscular mycorrhizal fungi and nitrogen addition on grassland temporal stability in the eastern Eurasian desert steppe

Abstract The temporal stability of grassland plant communities is substantially affected by soil nutrient enrichment. However, the potential main and interactive effects of arbuscular mycorrhizal fungi (AMF) and soil nitrogen (N) and phosphorus (P) enrichment on the stability of plant productivity have not yet been clarified. We combined a three‐year in situ field experiment to assess the impacts of soil fertilization and AMF on the stability of plant productivity. P addition decreased the stability of plant productivity by increasing the standard deviation relative to the mean of plant productivity. However, compared to species richness, the stability of C3 grasses and other functional groups asynchrony were the most important drivers changing the stability of plant productivity. The negative impacts of P addition overrode the impacts of AMF on the stability of plant productivity. Overall, our study suggests the importance of soil nutrient availability over AMF in terms of shaping the stability of plant productivity. Our results also suggest that three‐year anthropogenic soil nutrient enrichment could reduce the stability of plant communities in grassland regardless of AMF in the P‐limited grassland ecosystem.


| INTRODUC TI ON
Determining changes in the temporal stability (here defined as the mean/standard deviation of plant productivity over time; Tilman et al., 2006) of plant communities and the factors that regulate these changes is an essential aspect of ecological research Hautier et al., 2020). Terrestrial ecosystems are experiencing the effects of climate change (Lehmann & Rillig, 2014;Rillig Matthias et al., 2019;Song, Wan, et al., 2019;Song, Zong, et al., 2019) and intensive land use (Maestre et al., 2016(Maestre et al., , 2022Zhou, Luo, et al., 2019), which can change the stability of plant communities Hautier et al., 2020;Ives & Carpenter, 2007;Ma et al., 2017) and thus affect our ability to sustain current ecosystem functioning (Su et al., 2022;Tilman et al., 2014;Valencia et al., 2020).
Plant productivity in terrestrial ecosystems is usually limited by the availability of soil resources Eskelinen & Harrison, 2015;Xia & Wan, 2008), and chronic nutrient supply can benefit the production of plant biomass Xia & Wan, 2008). Soil nitrogen (N) and phosphorus (P) enrichments shape the stability of aboveground net primary productivity (ANPP) in grassland ecosystems Yang et al., 2022).
The global nutrient network experiment (NutNet), conducted in 34 grasslands over 7 years, revealed that N input reduces the stability of ANPP by greatly enhancing inter-annual variability of annual biomass production , and such N-induced negative effects have also been reported in other studies (Chen et al., 2021;Song, Wan, et al., 2019;Song, Zong, et al., 2019;Yang et al., 2012).
The effects of P addition on the stability of ANPP in grassland ecosystems can vary from negative Chen et al., 2021) to positive (Yang et al., 2014) by causing both greater variability and decreased variability in plant productivity Yang, Mariotte, et al., 2021;. Although previous field studies have found that the input of single nutrients (N or P) can strongly influence the stability of ANPP Chen et al., 2021), the interactive effects of N and P inputs on temporal stability in grassland ecosystems require further testing Chen et al., 2021).
Certain soil microorganisms, especially specific soil mutualists, are known to be important in temporally stabilizing plant communities, as shown in several field and greenhouse experiments Yang, 2021;Yang, Mariotte, et al., 2021;Yang, Shen, et al., 2018;Yang, Wagg, et al., 2018;. Arbuscular mycorrhizal fungi (AMF) are a type of fungus that forms a symbiotic relationship with host plants, and enhance the stability of plant productivity (Jia et al., 2022;Kang et al., 2020;Yang et al., 2014;Yang, Mariotte, et al., 2021;. Jia et al. (2021) reported that AMF can increase plant diversity (according to the Shannon-Wiener diversity index) and improve the stability of ANPP in an arid grassland, while the input of N had opposite effects in this environment.
Additionally, soil availability of N and P is known to alter the structure and function of AMF in grassland ecosystems (Han et al., 2020;Jiang et al., 2018;Treseder, 2004), potentially influencing the stabilizing effect of fungi on plant communities (Chen et al., 2021).
However, field evidence of the independent or interactive effects of AMF and soil enrichment with N and P on the stability of plant communities remains limited.
Previous in situ studies revealed that the stability of ANPP in grasslands could be modified by the richness of plant species , the asynchrony of plant species and/or functional groups (Huang et al., 2020;Sasaki et al., 2019;Xu et al., 2015) and the stability of plant populations and/or functional groups . Five-year-long study indicated that N input alone had a neutral impact on the stability of ANPP in meadow grassland (Kang et al., 2020). Plant species richness can weaken the effect of interannual variability on a plant community in terms of resistance/resilience enhancement that can come with greater species richness instead (Hautier et al., 2015). High plant species richness can enhance plant community resistance/resilience, which can then decrease inter-annual variability in productivity Yang, Mariotte, et al., 2021;. Thus, higher plant species richness in grassland ecosystems can increase the stability of ANPP due to asynchronous responses of plant species and/ or functional groups when environmental fluctuations occur (Huang et al., 2020). The asynchrony of plant functional groups, which is defined as different functional groups responding dissimilarly through time (Huang et al., 2020;Ma et al., 2022;Wilcox et al., 2017), is found as a vital driver of stability of plant productivity (Leps et al., 2019;Ma et al., 2022). Wilcox et al. (2017) found that plant asynchrony increased the stability of plant productivity across five continents.
The stability of specific plant population or functional groups is vital for stabilizing plant communities in grassland ecosystems Šmilauer et al., 2021). In a previous in situ experiment, , Yang, Mariotte, et al. (2021) and  showed that soil N enrichment and AMF independently alter the stability of ANPP by modifying the stability of dominant species but not plant species richness. Although the factors that determine the stability of plant communities are well-documented (Gross et al., 2014;Ives & Carpenter, 2007;Pimm, 1984), the relative importance of these driving factors in maintaining the stability of ANPP in response to soil nutrient enrichment and AMF remains poorly understood (Yang, Shen, et al., 2018;Yang, Wagg, et al., 2018).
The desert steppe within the eastern Eurasian steppe, which comprises more than 25% of the grassland area in China, is an exceedingly xeric grassland type (Kang et al., 2007). Previous studies found that plant production is extremely limited by N and P in this grassland ecosystem (Ma et al., 2020;Yang, Mariotte, et al., 2021;. Extreme soil nutrient limitation may hamper the mutualistic relationship between host plant and AMF, and then impairs the benefits of AMF for their host plants (Albornoz et al., 2021;Lambers et al., 2022). In the present study, we performed an in situ experiment to determine how AMF affects the stability of ANPP under N, P and N + P (combined) treatments in a desert steppe. We tested three hypotheses: (1) soil nutrient availability alone has a larger effect on plant community stability than AMF alone; (2) soil nutrient availability changes the stability of plant community through altering variability of plant productivity; (3) asynchrony across plant functional groups, as well as stability of dominant functional groups (e.g. C 3 grasses) have the greatest influence on overall plant community stability.

| Study site
Our in situ experiment site was located in Ningxia, Northwest China (37.7562° N, 107.2765° E; 1348 m asl). This region has a temperate continental climate with a mean annual temperature (MAT) of 8.1°C and a long-term mean annual precipitation (MAP) of 282.5 mm (from 1980 to 2020), of which >87.29% (~246.6 mm) occurs from 1 April to 30 September (Appendix S1). The type of soil in the region is light sierozem with a pH of 8.76, soil organic carbon concentration of 2.02 mg kg −1 and soil inorganic N and available plant P (Olsson-P) concentrations of 6.68 and 2.39 mg kg −1 , respectively.
The vegetation type is classified as temperate desert steppe, and the dominant plant functional group is C 3 grasses (mainly including Stipa breviflora and Agropyron mongolicum). Common plant species include Cleistogenes squarrosa (a perennial C 4 grass), Astragalus melilotoides (a perennial legume), Polygala tenuifolia, Euphorbia esula, Torularia humilis and Heteropappus altaicus (four types of perennial forbs) (Appendix S2). Except for Chenopodiaceae species, all plant species in our study site are colonized by AMF Yang, Mariotte, et al., 2021;. This region has been openly grazed since 2002 until it was fenced off to exclude livestock in 2017.

| Experimental design
The in situ experimental design was previously reported by , Yang, Mariotte, et al. (2021) and . Briefly, this study had a random block experimental design with five blocks and eight treatments, including a control (without N and P input), N input (N treatment; 10 g of N m −2 year −1 applied as ammonium nitrate), P input (P treatment; 10 g of P m −2 year −1 applied as superphosphate) and combined N and P input (N + P treatment; 10 g of N+ 10 g of P m −2 year −1 ) in no fungicide addition and fungicide addition plots. Therefore, eight experimental treatments were randomly assigned to eight experimental plots in each block.
Each experimental plot (2.2 m × 2.2 m) was surrounded by 1.0 m buffer walkways. The amounts of N and P applied exceeded the actual levels of atmospheric nutrient deposition in China but corresponded to the actual deposition of N and P in the agro-pasture ecotone in this region (Ma et al., 2020). Fungicide addition treatment was applied in each growing season using benomyl (6 g of effective constituent 10 L −1 of water m −2 applied per 2 weeks), while no fungicide addition treatment involved the application of equal amounts of water in each two-week period. Benomyl was used as a fungicide because it effectively reduces mycorrhizal root colonization of plants as well as extraradical hyphal density, but it has negligible effects on host plants and non-target soil bacteria, fungi and protozoa in grassland ecosystems (Jia et al., 2022;Yang, Mariotte, et al., 2021).

| Plant, soil and AM fungal traits
Two 0.5 m × 0.5 m quadrats were used randomly within each plot in mid-August (peak biomass period) in 2019-2021. The shoot biomass of each plant species in each plot was clipped to the soil surface, classified into plant species and then oven-dried at 65°C for nearly 72 h and weighed. We combined all shoot biomass of two quadrants in each experimental plot and defined ANPP (g m −2 ) as the total dry weights of each plant species. All plant species were also classified into plant functional groups (C 3 grasses, C 4 grasses, non-N 2 -fixing forbs and N 2 -fixing forbs; Appendix S2). ANPP data for plant species and functional groups in 2019 were previously reported Yang, Mariotte, et al., 2021;, whereas data for 2020 and 2021 are reported for the first time.
We randomly obtained three 10 cm deep soil cores in each plot in mid-August 2019-2021. We mixed soil samples and then sieved through a 2 mm mesh to separate the soil and the mixed roots, after which the soil samples were immediately frozen for neutral lipid fatty acids (NLFA) analysis (Andrino et al., 2021;Ngosong et al., 2012;Olsson & Lekberg, 2022). The inorganic N content of soil was analysed using a flow injection auto analyzer and the available P content of soil was analysed using the Olsen method (Chen et al., 2010). The mixed root samples in each plot were cut to a length of 0.5 cm and mixed, after which they were cleaned with 10% potassium hydroxide (KOH) at 90°C for 2 h, acidified in 2% hydrochloric acid (HCL) for 5 min and then stained with 0.05% trypan blue (Kormanik & Mcgraw, 1982). The colonization of mycorrhizal roots was then evaluated with a grid intersection method using a microscope (Giovannetti & Mosse, 1980).

| Assessment of temporal stability
Stability was calculated at the plant functional group and community level shoot biomass as the ratio of μ/σ, where μ is the mean shoot biomass of each functional group or total ANPP in each experimental plot and σ is the standard deviation (SD) of μ from 2019 to 2021 (Ma et al., 2017;Šmilauer et al., 2021). The asynchrony of the functional groups (1 − φ) was calculated as follows: where φ is plant functional group synchrony, σ 2 is the variance of the ANPP and σ i is the SD of the shoot biomass of the ith PFG in an experimental plot.

| Data analyses
To assess the impacts of N and P inputs and fungicide application on temporal stability at functional group and community levels and functional group asynchrony, three-way ANOVA with three fixed factors (N and P inputs and fungicide application) and one random factor (blocks) were conducted. Repeated measures ANOVA were conducted to evaluate the effects of N input, P inputs, fungicide application and their interaction on plant shoot biomass and diversity, soil available nutrients and AMF traits. The normality and homogeneity of variances were verified for all data using Kolmogorov-Smirnov and Levene tests, respectively. Three-way ANOVA and repeated measures ANOVA were performed using SPSS (version 22.0; IBM).
Structural equation modelling (SEM) was performed to determine how mycorrhizal suppression and N and P inputs altered the stability of the plant community. We use a χ 2 test, the Akaike information criterion (AIC) and the root mean square error (RMSE) of the approximation to test the fitness of SEM model. SEM was conducted using Amos (version 21; IBM).

| Effects of fungicide, N and P on plant community stability
Fungicide and P inputs interactively affected the stability of ANPP (F × P: F 1,28 = 5.70, p < .05; Table 1 and Figure 1a). Fungicide addition did not change the stability of ANPP in control plots; however, compared with P+ no-fungicide treatment, P+ fungicide significantly decreased the stability of ANPP by 91.53% under P input conditions ( Figure 1a). N and P inputs antagonistically affected the stability of ANPP (N × P: F 1,28 = 6.66, p < .05; Table 1 and Figure 1a). P addition significantly reduced the stability of ANPP in control, but it did not have this effect in N addition plots (Figure 1a). At the plant community level, fungicide addition significantly reduced functional groups asynchrony by 16.46% only in +P and +N+P plots (Figure 1b). At the plant functional group level, P input alone reduced the stability of C 3 grasses and non-N 2 -fixing forbs, but had no significant effect on that of C 4 grasses (Figure 1c-e). However, fungicide addition effectively reduced the stability of C 4 grasses across all fertilization treatments (Figure 1d). N and P inputs interactively altered the stability of N 2 -fixing forbs ( Figure 1f); P addition suppressed and enhanced the stability of N 2 -fixing forbs in the control and +N plots, respectively ( Figure 1f). P and N inputs alone significantly enhanced the temporal mean of ANPP (by 148.76% and 36.45%, respectively), whereas fungicide addition increased the temporal mean ANPP in +P and +N+P plots (Figure 2a). Fungicide addition also substantially increased the standard deviation of ANPP in P-addition plots ( Figure 2b).
According to the best-fitting SEM, AMF and P input directly affected the stability of ANPP and the negative effect of P input was stronger than the positive effect of AMF. Asynchrony of functional groups and the stability of C 3 grasses (i.e. the dominant functional group) were the best predictors of plant community stability, which was itself indirectly affected by the addition of P (Figure 3). The stability of N 2 -fixing forbs, non-N 2 -fixing forbs and C 4 grasses did not affect the stability of ANPP ( Figure 3). P addition also increased species richness, but this did not change the stability of ANPP (Figure 3).

| Effects of fungicide, N and P on plant, soil and AMF properties
Throughout the 3 years of the study, both N and P inputs signifi- (Appendix S6a-c), and P addition improved soil available P across all 3 years (Appendix S6d-f). Fungicide application alone did not alter soil available P and soil inorganic N in the present study (all p > .05; Appendix S6). In all 3 years, fungicide and P applications effectively TA B L E 1 Analysis of variance for the effects of fungicide application (F), nitrogen addition (N) and phosphorus addition (P) on the stability of plant functional group and plant community.

| Effects of N, P and fungicide inputs on the stability of ANPP
From the field study, the stability of plant community ANPP was substantially decreased by soil enrichment with N and P, which was in accordance with previous studies of different native F I G U R E 1 Response of temporal stability of plant community (a) and functional group asynchrony (b), and C 3 grasses (c), C 4 grasses (d), Non-N 2 -fixing forbs (e) and N 2 -fixing forbs (f) to nitrogen addition (N) and phosphorus addition (P) in no fungicide addition (dark green bars) and fungicide addition (light green bars) plots from 2019 to 2021. F, fungicide application. Data are presented as means + SE. *p < .05; **p < .01; NS p > .05.

F I G U R E 2
Response of the temporal mean (a) and standard deviation (b) of plant productivity to nitrogen addition (N) and phosphorus addition (P) in no fungicide addition (dark green bars) and fungicide addition (light green bars) plots from 2019 to 2020. F, fungicide application. Data are presented as means + SE. *p < .05; **p < .01; NS p > .05.
grassland ecosystems (Yang et al., 2012;Zhang et al., 2016) and a global net experiment . P addition affected the stability of ANPP mainly by increasing its standard deviation over its temporal mean in our field study. Compared to the first 2 years (2019 and 2020), P addition greatly increased ANPP by enhancing the shoot biomass of C 3 grasses in the third year (2021) and then lead to a disproportionate increase in standard deviation of ANPP in our field study. According to previous work, the negative impacts of P addition on plant community stability were also shown by the increase in standard deviation in a global nutrient network experiment over 7 years . In the present study, an antagonistic interaction between AMF and P input affected plant community stability. When P was not added, mycorrhizal suppression did not change plant community stability, which was comparable with that in no fungicide addition plots.
However, when P was added, it overrode the effects of AMF on plant community stability, presumably because AMF fungi markedly reduced the standard deviation of plant productivity in the P input plots. In accordance with a previous study (Yang et al., 2014), we found that AMF has the potential to enhance the stability of plant productivity under P input conditions. Carroll et al. (2022) found that N input had a destabilizing impact on plant community stability in grassland ecosystems across the world. However, in our field study, compared with P addition, N supply alone had a negligible effect on plant productivity and stability. Previous work found that sufficient precipitation in the growing season could improve the positive influence of N fertilizer on plant productivity in N-limited grassland (Lee et al., 2010). However, our study experienced a drought early in the growing season from 2020 to 2021, which could have limited the effects of N input on plant productivity in our study. Water limitation may hamper the effect of N input on plant productivity in our field study.
Plant diversity and/or the abundance of functional groups are known determinants of plant productivity stability (Huang et al., 2020;Ma et al., 2022;Zhou, Li, et al., 2019). We found that the stability of C 3 grass is a major contributor to plant community stability. Many of the C 3 grass species in our study site are relatively sensitive to soil P enrichment because the desert steppe is a P-impoverished ecosystem Yang, Mariotte, et al., 2021;. Our SEM model also indicated that functional group asynchrony was positively related to the stability of plant productivity. Specifically, P input increased plant functional groups asynchrony when AMF was intact, and the P addition treatments reduced the stability of C 3 grasses and non-N 2 -fixing forbs but stabilized that of C 4 grasses and N 2 -fixing forbs. This could explain the high degree of functional groups asynchrony observed in the present study. Notably, we found a synergistic interaction between P input and AMF, where AMF increased functional groups asynchrony under P input conditions. Different plant functional groups have varying mycorrhizal growth responses, in part because of the differential ability to supply carbon to AMF under P addition.
Functional groups in plant communities have different mycorrhizal growth responses and the ability of C supply for AMF under P input (Hoeksema et al., 2010;Lin et al., 2015). Specifically, C supply to AMF can be reduced for certain functional groups under P input, causing asynchrony in ANPP across functional groups. Thus, the interaction between soil nutrient availability and AMF may alter the stability of plant productivity, particularly in arid grassland ecosystems.

| Effects of N, P and fungicide inputs on environmental variables
Previous studies have revealed that soil nutrient enrichment alters plant species richness, thereby changing the stability of plant productivity (Hautier et al., 2015;Huang et al., 2020;Xu et al., 2021;Yang et al., 2012). Zhou et al. (2020) also showed that a positive relationship exists between plant productivity stability and plant species richness. However, in the present study, we found no positive diversity-stability relationship. This may have been due to the low variation in plant species richness (an increase of only 1-3 plant species) in the P input plots. Moreover, two N 2 fixing forbs, Oxytropis racemosa and Gueldenstaedtia verna, dominated the P input plots, but accounted for a low proportion of ANPP and had lower stability than other functional groups. Similarly, previous field experiments have found that plant species richness is not the primary driver of stability of ANPP (Ma et al., 2021;Xu et al., 2015).
We showed that P input and fungicide application suppressed mycorrhizal root colonization and its NLFA concentration in 2019-2021, which aligns with previous work in native grassland ecosystems (Qiao et al., 2019;Yang, Shen, et al., 2018;Yang, Wagg, et al., 2018). Furthermore, the application of benomyl did not alter the NLFA concentrations of other microorganism groups (e.g. pathogenic fungi) in the present study, confirming that this fungicide effectively suppresses the structure of AMF without affecting other microorganisms, which is consistent with previous field studies conducted in a temperate steppe (Yang et al., 2014) and desert steppe Yang, Mariotte, et al., 2021;.
Our in situ study revealed that the negative effects of P addition outweigh the effects of AMF and N input on plant community stability in this P-impoverished grassland ecosystem. Yang et al. (2014) found that P supplementation tended to increase plant productivity stability when AMF was intact; however, our field study revealed that the addition of P had the opposite effect, and this was supported by the results of global NutNet study .
The different responses of plant stability to soil N and P enrichment and AMF suggest that these factors may have interacting effects on plant productivity stability. However, our in situ field study lasted only 3 years, a relatively short duration for analyzing plant stability . Understanding of plant stability to long-term fertilizer and fungicide input is scarce , and requires further study.

| CON CLUS IONS
In conclusion, our study indicated that P inputs decreased plant productivity stability by increasing the standard deviation of plant productivity directly and decreasing the stability of the dominant functional group (C 3 grasses). Furthermore, we found that AMF have F I G U R E 5 Response of species richness (a-c), Shannon diversity index (d-f) to nitrogen addition (N) and phosphorus addition (P) in no fungicide addition (dark green bars) and fungicide addition (light green bars) plots from 2019 to 2020. Data presented as means + SE. *p < .05; **p < .01; NS p > .05.
potential effects on plant productivity stability associated with P input in an arid grassland ecosystem. The negative effects of P input overrode the effects of AMF and N input on plant community stability. Soil fertility and AMF are important for the stability of ANPP in grassland ecosystems; therefore, our findings improve our understanding of the response of plant community dynamics to AMF and anthropogenic soil nutrient enrichment.

ACK N O WLE D G E M ENTS
We thank

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare no conflict of interest.